State of the art paper Cardiology On the present and future role of Lp-PLA2 in atherosclerosis-related cardiovascular risk prediction and management Zlatko Fras1,2, Jure Tršan1,3, Maciej Banach4,5 1Centre for Preventive Cardiology, Department of Vascular Medicine, Corresponding author: Division of Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia Prof. Zlatko Fras MD, PhD, 2Chair of Internal Medicine, Medical Faculty, University of Ljubljana, Ljubljana, FESC, FACC Slovenia Centre for 3Medical Faculty, University of Ljubljana, Ljubljana, Slovenia Preventive Cardiology 4Department of Hypertension, Medical University of Lodz, Poland Department of 5Polish Mother’s Memorial Hospital Research Institute, Lodz, Poland Vascular Medicine Division of Medicine Submitted: 10 January 2020; Accepted: 2 February 2020 University Medical Online publication: 20 August 2020 Centre Ljubljana Zaloška 7 Arch Med Sci 2021; 17 (4): 954–964 SI-1525 Ljubljana, Slovenia DOI: https://doi.org/10.5114/aoms.2020.98195 Medical Faculty Copyright © 2020 Termedia & Banach University of Ljubljana Vrazov trg 2 SI-1000 Ljubljana, Slovenia Abstract Phone: +386-1-522-31-52 E-mail: [email protected] Circulating concentration and activity of secretory phospholipase A2 (sPLA2) and lipoprotein-associated phospholipase A2 (Lp-PLA2) have been proven as biomarkers of increased risk of atherosclerosis-related cardiovascular dis- ease (ASCVD). Lp-PLA2 might be part of the atherosclerotic process and may contribute to plaque destabilisation through inflammatory activity within atherosclerotic lesions. However, all attempts to translate the inhibition of phospholipase into clinically beneficial ASCVD risk reduction, including in randomised studies, by either non-specific inhibition of sPLA2 (by varesp- ladib) or specific Lp-PLA2 inhibition by darapladib, unexpectedly failed. This gives us a strong imperative to continue research aimed at a better under- standing of how Lp-PLA2 and sPLA2 regulate vascular inflammation and ath- erosclerotic plaque development. From the clinical viewpoint there is a need to establish and validate the existing and emerging novel anti-inflammatory therapeutic strategies to fight against ASCVD development, by using poten- tially better animal models and differently designed clinical trials in humans. Key words: atherogenesis, phospholipases, biomarker, secretory phospholipases A2, lipoprotein-associated phospholipase A2 (Lp-PLA2), prognosis, anti-inflammatory agents. Introduction – the A2 group phospholipases (PLA2s) The phospholipases are enzymes that hydrolyse phospholipids. They are classified into different groups by their molecular weight, their cata- lytic residues, and their dependence (or lack thereof) on calcium [1]. The A2 group of phospholipases specifically hydrolyse the ester bond of the fatty acid at the sn-2 position of the glycerophospholipids and, by doing so, release both fatty acids and lysophospholipids [1, 2]. Secretory PLA2 (sPLA2) are calcium-dependent, low-molecular-weight enzymes that include different groups, named I-III, V, and IX-XIV. Also cy- tosolic PLA2 (cPLA2, GIV) are calcium dependent. On the other hand, cal- cium independent groups are GV PLA2 (iPLA2) and lipoprotein-associated phospholipase A2 (Lp-PLA2 or, as it is also called, platelet-activating fac- Creative Commons licenses: This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY -NC -SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/). On the present and future role of Lp-PLA2 in atherosclerosis-related cardiovascular risk prediction and management tor acetylhydrolases; PAF-AH, GVII/GVIII). We also Phosphatidylcholine hydrolysis by sPLA2 results in know of lysosomal PLA2 (GXV) and adipose-specif- very-low density lipoprotein (VLDL) and low densi- ic phospholipase A2 (AdPLA2, GXVI) [1, 3–6]. ty lipoprotein (LDL) particles with altered confor- mation of apolipoprotein B (apoB). These processes sPLA2 family includes 12 isoforms, and despite sharing some common features they are functional- result in smaller, denser, and more electronegative ly distinct proteins with specific tissue distributions lipoprotein particles [1, 3, 5] that are less avidly in- [7–9] and enzymatic properties [9]. They hydrolyse ternalised by the hepatic apoB/E (LDL) receptor [3, phospholipids from the surface of cell membranes, 11], with a prolonged residence time in the circula- native lipoproteins, and oxidatively-modified lipo- tion and further susceptibility to oxidation [3]. GV proteins to produce many different bioactive lipids and GX sPLA2 enzymes hydrolyse phosphatidylcho- that include arachidonic acid (and consequently line on the surface of VLDL and LDL at least 20-fold also eicosanoids – prostaglandins, thromboxanes, more efficiently than GIIA sPLA2 and as such have leukotrienes), non-esterified fatty acids, lyso- a potential to act extracellularly [1, 3, 12–14]. On phospholipids, lyso-platelet acting factor, and oxi- the other hand, GIIA sPLA2 shows enhanced ability dised non-esterified fatty acids [3, 8]. In contrast, to hydrolyse oxidised LDL and acts within intima and macrophages [1, 3]. Hydrolysis of phospho- Lp-PLA2 requires oxidised phospholipids as a sub- strate (platelet-activating factor (PAF), PAF-like sub- lipids on high-density lipoprotein (HDL) results in stances and oxidised phospholipids) [3, 10]. impaired cholesterol-efflux capacity – the ability of Circulating concentration and enzymatic activi- HDL to accept cholesterol from macrophages [3, 5, ty of secretory phospholipase A (sPLA ) and lipo- 15]. The conformational changes in apoB also in- 2 2 crease intimal proteoglycan binding [1, 3, 16–18] protein-associated phospholipase A2 (Lp-PLA2) have been evaluated as biomarkers of cardiovas- and therefore promote retention of these athero- cular risk in populations of apparently healthy in- genic lipoproteins and cholesterol crystal precipita- tion [19]. sPLA -mediated phospholipid hydrolysis dividuals, as well as in patients with established 2 further increases its vasoactive, chemotactic, and coronary heart disease (CHD) [1, 3–6]. proinflammatory role [3, 5] because it increases On the role of PLA s in atherogenesis oxidative stress through generation of arachidon- 2 ic acid (including eicosanoids), lysophospholipids, Secretory phospholipase A2 (sPLA2) and non-esterified fatty acids [3, 5, 20]. and atherosclerosis Lipoprotein-associated phospholipase A Six isoforms of the sPLA family are described to 2 2 and atherosclerosis be present in atherosclerotic lesions: IIA, IID, IIE, III, V, and X, and they have been reported to have a po- Lp-PLA2 is secreted primarily by macrophages tential causal role in atherogenesis [3, 5] (Figure 1). and by some other inflammatory and non-inflam- Varespladib Oxidized Native LDL phospholipids Lp-PLA sPLA 2 Monocytes, 2 secretion lymphocytes + Small, Adhesion dense LDL LUMEN molecules INTIMA sPLA Atheroma + + + Varespladib 2 Endothelial Chemotaxis + dysfunction + + + LPC, ox NEFA Lp-PLA2 Cytokine activation sPLA + Macrophage Foam cell 2 Cell apoptosis oxLDL Necrotic core sPLA Lp-PLA2 2 + Darapladib growth Apoptosis Varespladib cytotoxic effects Darapladib Smooth muscle cells Migration, apoptosis MEDIA Figure 1. Schematic presentation of the roles of sPLA2 and Lp-PLA2 in atherogenesis, as well as the potential sites for therapeutic inhibition, by using either sPLA2 non-specific (e.g. varespladib), or a specific Lp-PLA2 inhibitor (darapladib) sPLA2 – secretory phospholipase A2, Lp-PLA2 – lipoprotein-associated phospholipase A2, LPC – lysophosphatidylcholine, oxNEFA – oxidised non-esterified fatty acids, oxLDL – oxidised low-density lipoprotein particle. Arch Med Sci 4, June / 2021 955 Zlatko Fras, Jure Tršan, Maciej Banach matory cells involved in atherogenesis [21] (Fig- ably because it only detects a reduced percentage ure 1). In plasma, Lp-PLA2 circulates in active form of total Lp-PLA2 that is not in interaction with lipo- as a complex with LDL (80–85%), HDL (15–20%), protein [25, 26]. The Food and Drug Administra- and, to a lesser extent, Lp(a) [10, 21]. tion (FDA) approved the PLAC® Test for measuring ® Endothelial dysfunction is a well-established the Lp-PLA2 mass concentration (2003) and PLAC vascular response to cardiovascular risk factors, Test Activity (2014) for enzymatic activity in order which precedes the development of atherosclero- to improve diagnostics and prediction of ASCVD sis and is involved in the promotion of both the in clinical practice [26, 27]. The performance of early and late mechanisms of progression [22]. It the PLAC® Test is superior to other alternative is characterised by the expression of more adhe- commercially available tests [27]. It is a standard sion molecules and increased endothelial permea- indirect ELISA immunoassay that uses two mono- bility; hence, LDL particles can transmigrate more clonal antibodies: a primary antibody to bind to easily to arterial intima [10, 22]. Because of the Lp-PLA2 enzyme from the blood sample and an reduced content of antioxidants in arterial intima enzyme-conjugated secondary antibody to de- LDL particles are exposed to further oxidation. ® tect it. The PLAC Test Activity uses the Lp-PLA2 Consequently, the Lp-PLA2 is activated by an abun- to hydrolyse the sn-2 position of the substrate, dance of oxidised phospholipids present in OxLDL 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphati- [10]. As such, Lp-PLA2-driven hydrolysis of the ox-